Use of IL-18 inhibitors for treating head injuries

ABSTRACT

The invention relates to the use of inhibitors of IL-18 in the preparation of a medicament for treatment and/or prevention of central nervous system injury, in particular of traumatic head injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase Entry Under 35 U.S.C. 371 ofInternational Application No. PCT/EP02/05666, filed May 23, 2002 whichdesignated the U.S.

FIELD OF THE INVENTION

The present invention is in the field of pathological conditions of thebrain. More specifically, it relates to the use of an inhibitor of IL-18for the treatment and/or prevention of central nervous system (CNS)injury, in particular traumatic brain injury.

BACKGROUND OF THE INVENTION

In 1989, an endotoxin-induced serum activity that induced interferon-γ(IFN-γ) obtained from mouse spleen cells was described (Nakamura et al.,1989). This serum activity functioned not as a direct inducer of IFN-γbut rather as a co-stimulant together with IL-2 or mitogens. An attemptto purify the activity from post-endotoxin mouse serum revealed anapparently homogeneous 50-55 kDa protein. Since other cytokines can actas co-stimulants for IFN-γ production, the failure of neutralizingantibodies to IL-1, IL4, IL-5, IL-6, or TNF to neutralize the serumactivity suggested it was a distinct factor. In 1995, the samescientists demonstrated that the endotoxin-induced co-stimulant forIFN-γ production was present in extracts of livers from micepreconditioned with P. acnes (Okamura et al., 1995). In this model, thehepatic macrophage population (Kupffer cells) expand and in these mice,a low dose of bacterial lipopolysaccharide (LPS), which innon-preconditioned mice is not lethal, becomes lethal. The factor, namedIFN-γ-inducing factor (IGIF) and later designated interleukin-18(IL-18), was purified to homogeneity from 1,200 grams of P.acnes-treated mouse livers. Degenerate oligonucleotides derived fromamino acid sequences of purified IL-18 were used to clone a murine IL-18cDNA. IL-18 is an 18-19 kDa protein of 157 amino acids, which has noobvious similarities to any peptide in the databases. Messenger RNAs forIL-18 and interleukin-12 (IL-12) are readily detected in Kupffer cellsand activated macrophages. Recombinant IL-18 induces IFN-gamma morepotently than does IL-12, apparently through a separate pathway(Micallef et al., 1996). Similar to the endotoxin-induced serumactivity, IL-18 does not induce IFN-γ by itself, but functions primarilyas a co-stimulant with mitogens or IL-2. IL-18 enhances T cellproliferation, apparently through an IL-2-dependent pathway, andenhances Th1 cytokine production in vitro and exhibits synergism whencombined with IL-12 in terms of enhanced IFN-γ production (Maliszewskiet al., 1990).

After the murine form was cloned, the human cDNA sequence for IL-18 wasreported in 1996 (Ushio et al., 1996).

By cloning IL-18 from affected tissues and studying IL-18 geneexpression, a close association of this cytokine with an autoimmunedisease was found. The non-obese diabetic (NOD) mouse spontaneouslydevelops autoimmune insulitis and diabetes, which can be accelerated andsynchronized by a single injection of cyclophosphamide. IL-18 mRNA wasdemonstrated by reverse transcriptase PCR in NOD mouse pancreas duringearly stages of insulitis. Levels of IL-18 mRNA increased rapidly aftercyclophosphamide treatment and preceded a rise in IFN-γ mRNA, andsubsequently diabetes. Interestingly, these kinetics mimic that ofIL-12-p40 mRNA, resulting in a close correlation of individual mRNAlevels. Cloning of the IL-18 cDNA from pancreas RNA followed bysequencing revealed identity with the IL-18 sequence cloned from Kupffercells and in vivo pre-activated macrophages. Also NOD mouse macrophagesresponded to cyclophosphamide with IL-18 gene expression whilemacrophages from Balb/c mice treated in parallel did not. Therefore,IL-18 expression is abnormally regulated in autoimmune NOD mice andclosely associated with diabetes development (Rothe et al., 1997).

IL-18 plays a potential role in immunoregulation or in inflammation byaugmenting the functional activity of Fas ligand on Th1 cells (Conti etal., 1997). IL-18 is also expressed in the adrenal cortex and thereforemight be a secreted neuro-immunomodulator, playing an important role inorchestrating the immune system following a stressful experience(Chater, 1986).

In vivo, IL-18 is formed by cleavage of pro-IL-18, and its endogenousactivity appears to account for IFN-γ production in P. acnes andLPS-mediated lethality. Mature IL-18 is produced from its precursor bythe IL-1β converting enzyme (IL-1beta-converting enzyme, ICE,caspase-1).

The IL-18 receptor consists of at least two components, co-operating inligand binding. High- and low-affinity binding sites for IL-18 werefound in murine IL-12 stimulated T cells (Yoshimoto et al., 1998),suggesting a multiple chain receptor complex. Two receptor subunits havebeen identified so far, both belonging to the IL-1 receptor family(Parnet et al., 1996). The signal transduction of IL-18 involvesactivation of NF-κB (DiDonato et al., 1997).

Recently, a soluble protein having a high affinity for IL-18 has beenisolated from human urine, and the human and mouse cDNAs were described(Novick et al., 1999; WO 99/09063). The protein has been designatedIL-18 binding protein (IL-18BP).

IL-18BP is not the extracellular domain of one of the known IL18receptors, but a secreted, naturally circulating protein. It belongs toa novel family of secreted proteins. The family further includes severalPoxvirus-encoded proteins which have a high homology to IL-18BP (Novicket al., 1999). IL-18BP is constitutively expressed in the spleen,belongs to the immunoglobulin superfamily, and has limited homology tothe IL-1 type II receptor. Its gene was localized on human chromosome11q13, and no exon coding for a transmembrane domain was found in an 8.3kb genomic sequence (Novick et al., 1999).

Four human and two mouse isoforms of IL-18BP, resulting from mRNAsplicing and found in various cDNA libraries and have been expressed,purified, and assessed for binding and neutralization of IL-18biological activities (Kim et al., 2000). Human IL-18BP isoform a(IL-18BPa) exhibited the greatest affinity for IL-18 with a rapidon-rate, a slow off-rate, and a dissociation constant (K(d)) of 399 pM.IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminalamino acids; the K(d) of IL-18BPc is 10-fold less (2.94 nM).Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18>95% at a molarexcess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domainand lack the ability to bind or neutralize IL-18. Murine IL-18BPc andIL-18BPd isoforms, possessing the identical Ig domain, alsoneutralize >95% murine IL-18 at a molar excess of two. However, murineIL-18BPd, which shares a common C-terminal motif with human IL-18BPa,also neutralizes human IL-18. Molecular modelling identified a largemixed electrostatic and hydrophobic binding site in the Ig domain ofIL-18BP, which could account for its high affinity binding to the ligand(Kim et al., 2000).

Traumatic brain injury (TBI), also simply called head injury, or closedhead injury (CHI), refers to an injury of the central nervous systemwhere there is damage to the brain caused by an external blow to thehead. It mostly happens during car or bicycle accidents, but may alsooccur as the result of near drowning, heart attack, stroke andinfections. This type of traumatic brain injury would usually result dueto the lack of oxygen or blood supply to the brain, and therefore can bereferred to as an “anoxic injury”.

Closed head injury occurs when there is a blow to the head as in a motorvehicle accident or a fall. In this case, the skull hits a stationaryobject and the brain, which is inside the skull, turns and twists on itsaxis (the brain stem), causing localized or widespread damage. Also, thebrain, a soft mass surrounded by fluid that allows it to “float,” mayrebound against the skull resulting in further damage.

There may be a period of unconsciousness immediately following thetrauma, which may last minutes, weeks or months. Due to the twisting andrebounding, the traumatically brain injured patient usually receivesdamage or bruising to many parts of the brain. This is called diffusedamage, or “non-missile injury” to the brain. The types of brain damagesoccurring in non-missile injuries may be classified as either primary orsecondary.

Primary brain damage occurs at the time of injury, mainly at the sitesof impact, in particular when a skull fraction is present. Largecontusions may be associated with an intracerebral haemorrhage, oraccompanied by cortical lacerations. Diffuse axonal injuries occur as aresult of shearing and tensile strains of neuronal processes produced byrotational movements of the brain within the skull. There may be smallheamorrhagic lesions or diffuse damage to axons, which can only bedetected microscopically.

Secondary brain damage occurs as a result of complications developingafter the moment of injury. They include intracranial hemorrhage,traumatic damage to extracerebral arteries, intracranial herniation,hypoxic brain damage or meningitis.

An “open head injury” is a visible assault to the head and may resultfrom a gunshot wound, an accident or an object going through the skullinto the brain (“missile injury to the brain”), This type of head injuryis more likely to damage a specific area of the brain.

So called “mild brain injury” may occur with no loss of consciousnessand possibly only a dazed feeling or confused state lasting a shorttime. Although medical care administered may be minimal, persons withbrain injury without coma may experience symptoms and impairmentssimilar to those suffered by the survivor of a coma injury.

In response to the trauma, changes occur in the brain, which requiremonitoring to prevent further damage. The brain's size frequentlyincreases after a severe head injury. This is called brain swelling andoccurs when there is an increase in the amount of blood to the brain.Later in the illness water may collect in the brain, which is calledbrain edema. Both brain swelling and brain edema result in excessivepressure in the brain called intracranial pressure (“ICP”).

Coma is the prolonged period of unconsciousness immediately followingthe traumatic head injury.

There are several levels of coma. Coma levels can be measured by theprogression of responsiveness of the head injured person. In the acutephase of head injury the “Glasgow Coma Scale” is used. As the patientimproves or stabilizes, the “Rancho Los Amigos Scale” is used whichmeasures levels of cognitive (understanding and reasoning) thinking.

Brain injury frequently results in persisting debility, such aspost-traumatic epilepsy, persistent vegetative state, or post-traumaticdementia.

Spinal cord injury is another type of CNS injury. Spinal cord injuriesaccount for the majority of hospital admissions for paraplegia andtetraplegia. Over 80% occur as a result of road accidents. Two maingroups of injury are recognized clinically: open injuries and closedinjuries.

Open injuries cause direct trauma of the spinal cord and nerve roots.Perforating injuries can cause extensive disruption and haemorrhage.Closed injuries account for most spinal injuries and are usuallyassociated with a fracture/dislocation of the spinal column, which isusually demonstrable radiologically. Damage to the cord depends on theextent of the bony injuries and can be considered in two main stages:Primary damage, which are contusions, nerve fibre transections andheamorrhagic necrosis, and secondary damage, which are extraduralhaematoma, infarction, infection and oedema.

Late effects of cord damage include: ascending and descendinganterograde degeneration of damaged nerve fibers, post-traumaticsyringomelyia, and systemic effects of paraplegia, such as urinary tractand chest infections, pressure sores and muscle wasting.

The pathology of traumatic brain injury is very complex and still poorlyunderstood. Research efforts in the past decade have highlighted animportant role of cytokines released systemically and locally within theintrathecal compartment after brain injury, and a dual effect ofpro-inflammatory cytokines, such as TNF, IL-6, or IL-8, was hypothesizedbased on findings of time-dependent beneficial and adverse effects ofthese mediators (Morganti-Kossmann et al., 1997; Kossmann et al., 1997;Shohami et al., 1999, Scherbel et al., 1999; Whalen et al., 2000). Asdescribed above, a recently discovered cytokine of the IL-1 family isIL-18. Recent studies have demonstrated that IL-18 is constitutivelyexpressed in the CNS of mice, rats, and humans in vivo (Culhane et al.,1998; Jander and Stoll, 1998; Prinz et al., 1999; Fassbender et al.,1999; Wheeler et al., 2000), as well as in primary cultures ofastrocytes and microglia, but not neurons, in vitro (Conti et al.,1999). Increased IL-18 levels were detected in the cerebrospinal fluid(CSF) of patients with inflammatory CNS diseases, such as bacterialmeningitis and viral meningoencephalitis, but not in the CSF of multiplesclerosis (MS) patients (Fassbender et al., 1999). In contrast to thefinding of generally low intrathecal IL-18 levels in MS patients,increased IL-18 mRNA expression was demonstrated in spinal cords ofLewis rats with experimental autoimmune encephalomyelitis (EAE), theanimal model for MS (Jander and Stoll, 1998). The expression andfunctional significance of IL-18 in neurotrauma has not beeninvestigated until now.

SUMMARY OF THE INVENTION

The present invention relates to the pathophysiological role of IL-18 inCNS diseases. It is based on the finding that the treatment of mice withinhibitors of IL-18, either one hour or three days after experimentalclosed head injury (CHI), results in an improved recovery and attenuatedextent of brain damage as compared to control animals. The inventiontherefore relates to the use of an IL-18 inhibitor for the manufactureof a medicament for treatment and/or prevention of central nervoussystem (CNS) injury, and in particular of traumatic brain injury.

The use of combinations of an IL-18 inhibitor with an interferon and/orTNF and/or inhibitors of inflammation and/or anfioxidants are alsoprovided according to the invention. In order to apply gene therapeuticapproaches to deliver the IL-18 inhibitor to diseased tissues or cells,further aspects of the invention relate to the use of nucleic acidmolecules comprising the coding sequence of an IL-18 inhibitor for thetreatment and/or prevention of the CNS injury. The invention alsorelates to the use of cells genetically engineered to express IL-18inhibitors for the prevention and/or treatment of CNS injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a histogram depicting the serum levels of intracerebralIL-18 (ng/ml) in whole brain (hatched), in the left hemisphere (black)or in the right hemisphere (gray) under different conditions.

FIG. 2 shows the development of NSS (Neurological Severity Score)measured 1 hour (h), 24 h, 72 h or 168 h following trauma, either with50 μg of IL-18BP administered i.p. at 1 h following trauma (squares), orwith injection of vehicle only (control, circles).

FIG. 3 shows the ΔNSS measured 24 h, 72 h or 168 h following trauma,either with 50 μg of IL-18BP administered i.p. at 1 h following trauma(squares), or with injection of vehicle only (control, circles).

FIG. 4 shows the ΔNSS measured 1 h, 24 h, 3 days (d), 7d or 14dfollowing trauma, either with 50 μg of IL-18BP administered i.p. eitheras a single dose at 3d following trauma (diamonds), or with a doubledose at 1 h and 3d following trauma (squares) or injection of vehicleonly (control, triangle).

DESCRIPTION OF THE INVENTION

The present invention is based on the finding of a statisticallysignificant beneficial effect of an IL-18 inhibitor on the recovery frombrain injury in a murine model of closed head injury. In accordance withthe present invention, it has further been found that IL-18 isup-regulated in the brain and cerebrospinal fluid after traumatic braininjury, indicating that this pro-inflammatory cytokine plays animportant role in the pathogenesis of brain injury.

Therefore, the invention relates to the use of an IL-18 inhibitor forthe manufacture of a medicament for treatment and/or prevention ofcentral nervous system (CNS) injury.

The invention further relates to the use of an IL-18 inhibitor for themanufacture of a medicament for treatment and/or prevention ofcomplications and late effects of CNS injury.

In preferred embodiments of the invention, the CNS injury is traumaticbrain injury or closed head injury.

In a further preferred embodiment, the CNS injury is spinal cord injury.

In yet a further yet a further preferred embodiment of the invention,the brain injury is of vascular origin.

Within the context of the present invention, the expression “centralnervous system injury” or “CNS injury” relates to any injury to thebrain or spinal cord, regardless of the age at onset, or the underlyingcause. The underlying cause may e.g. be mechanical, or an infection. CNSinjury and its clinical symptoms and implications have been described indetail in the “Background of the invention”. CNS injury includes e.g.trauma, or any other damage of the brain or spinal cord, and it may alsobe called neurotrauma.

Brain injury may for example include or result in any one, or more, ofthe following: 1. Attention impairment; 2. cognition impairment; 3.language impairment; 4. memory impairment; 5. conduct disorder; 6. motordisorder; 6. any other neurological dysfunction.

Spinal cord injury may for example result in paraplegia or tetraplegia.

Complications or late effects of CNS injury may also be treated and/orprevented in accordance with the present invention. Complications andlate effects of brain injuries have been described above in the“Background of the invention”. They include, but are not limited to,coma, meningitis, post-traumatic epilepsy, post-traumatic dementia,degeneration of nerve fibers, or post-traumatic syringomyelia, orhemorrhage, for example.

The present invention also relates to the use of IL-18 inhibitors forthe preparation of a medicament for treatment and/or prevention of anyinjury to the brain that is vascular in origin, such as hypoxic braindamage with cerebral infarction, ischemia, cerebrovascular accident, orstroke.

The terms “treating” and “preventing”, as used herein, should beunderstood as partially or totally preventing, inhibiting, attenuating,ameliorating or reversing one or more symptoms or cause(s) of CNSinjury, as well as symptoms, diseases or complications accompanying CNSinjury. When “treating” CNS injury, the substances according to theinvention are given after onset of the disease, “prevention” relates toadministration of the substances before signs of disease can be noted inthe patient.

Treatment of CNS injury is preferred in accordance with the presentinvention. Preferably, in order to treat CNS injury, the IL-18 inhibitoris administered as soon as possible after CNS injury, e.g. within thefirst hour after the injury. However, as shown in the Examples below,one IL-18 inhibitor was shown to exert its beneficial effect on braininjury even when administered three days after brain injury occurred.Therefore, in order to treat CNS injury, it is preferred to administerthe IL-18 inhibitor within three days from the injury.

The term “inhibitor of IL-18” within the context of this invention,refers to any molecule modulating IL-18 production and/or action in sucha way that IL-18 production and/or action is attenuated, reduced, orpartially, substantially or completely prevented or blocked. The term“IL-18 inhibitor” is meant to encompass inhibitors of IL-18 production,as well as of inhibitors of IL-18 action.

An inhibitor of production can be any molecule negatively affecting thesynthesis, processing or maturation of IL-18. The inhibitors consideredaccording to the invention can be, for example, suppressors of geneexpression of the interleukin IL-18, antisense mRNAs reducing orpreventing the transcription of the IL-18 mRNA or leading to degradationof the mRNA, proteins impairing correct folding, or partially orsubstantially preventing secretion of IL-18, proteases degrading IL-18,once it has been synthesized, inhibitors of proteases cleaving pro-IL-18in order to generate mature IL-18, such as inhibitors of caspase-1, andthe like.

An inhibitor of IL-18 action can be an IL-18 antagonist, for example.Antagonists can either bind to or sequester the IL-18 molecule itselfwith sufficient affinity and specificity to partially or substantiallyneutralize the IL-18 or IL-18 binding site(s) responsible for IL-18binding to its ligands (like, e.g. to its receptors). An antagonist mayalso inhibit the IL-18 signaling pathway, which is activated within thecells upon IL-18 binding to its receptor.

Inhibitors of IL-18 action may also be soluble IL-18 receptors ormolecules mimicking the receptors, or agents blocking the IL-18receptors, or IL-18 antibodies, such as polyclonal or monoclonalantibodies, or any other agent or molecule preventing the binding ofIL-18 to its targets, thus diminishing or preventing triggering of theintra- or extracellular reactions mediated by IL-18.

In a preferred embodiment of the present invention, the inhibitor ofIL-18 is selected from inhibitors of caspase-1 (ICE), antibodiesdirected against IL-18, antibodies directed against any of the IL-18receptor subunits, inhibitors of the IL-18 signaling pathway,antagonists of IL-18 which compete with IL-18, or bind to, and block theIL-18 receptor, and IL-18 binding proteins, isoforms, muteins, fusedproteins, functional derivatives, active fractions or circularlypermutated derivatives, or salts thereof.

The term “IL-18 binding proteins” is used herein synonymously with“IL18BP”. It comprises IL-18 binding proteins as defined in WO 99/09063or in Novick et al., 1999, including splice variants and/or isoforms ofIL-18 binding proteins, as defined in Kim et al., 2000. In particular,human isoforms a and c of IL-18BP are useful in accordance with thepresence invention. The proteins useful according to the presentinvention may be glycosylated or non-glycosylated, they may be derivedfrom natural sources, such as urine, or they may preferably be producedrecombinantly. Recombinant expression may be carried out in prokaryoticexpression systems like E. coli, or in eukaryotic, and preferably inmammalian, expression systems. A cell line particularly well suited forthe IL-18 inhibitors of the present invention is the Chinese hamsterovary (CHO) cell.

Recombinant production of the IL-18 inhibitor, when recombinantlyexpressed in mammalian cells or cell lines, may preferably be carriedout in serum free cell culture medium.

As used herein the term “muteins” refers to analogs of an IL-18BP, oranalogs of a viral IL-18BP, in which one or more of the amino acidresidues of a natural IL-18BP or viral IL-18BP are replaced by differentamino acid residues, or are deleted, or one or more amino acid residuesare added to the natural sequence of an IL-18BP, or a viral IL-18BP,without reducing considerably the activity of the resulting products ascompared with the wild type IL-18BP or viral IL-18BP. These muteins areprepared by known synthesis and/or by site-directed mutagenesistechniques, or any other known technique suitable therefor.

Any such mutein preferably has a sequence of amino acids sufficientlyduplicative of that of an IL-18BP, or sufficiently duplicative of aviral IL-18BP, such as to have substantially similar activity toIL-18BP. One activity of IL-18BP is its capability of binding IL-18. Aslong as the mutein has substantial binding activity to IL-18, it can beused in the purification of IL-18, such as by means of affinitychromatography, and thus can be considered to have substantially similaractivity to IL-18BP. Thus, it can be determined whether any given muteinhas an activity substantially similar to IL-18BP by means of routineexperimentation comprising subjecting such a mutein, e.g., to a simplesandwich competition assay to determine whether or not it binds to anappropriately labeled IL-18, such as radioimmunoassay or ELISA assay.Simple functional assays for assessing the biological activity ofIL-18BP were described in detail in WO 99/09063, e.g. in examples 2(binding to IL-18 as assessed by cross-linking) or 5 (inhibition ofIL-18 induced INF-gamma induction in mononuclear blood cells).

In a preferred embodiment, any such mutein has at least 40% identity orhomology with the sequence of either an IL-18BP or a virally-encodedIL-18BP homologue. More preferably, it has at least 50%, at least 60%,at least 70%, at least 80% or, most preferably, at least 90% identity orhomology thereto.

Muteins of IL-18BP polypeptides or muteins of viral IL-18BPs, which canbe used in accordance with the present invention, or nucleic acid codingtherefor, include a finite set of substantially corresponding sequencesas substitution peptides or polynucleotides which can be routinelyobtained by one of ordinary skill in the art, without undueexperimentation, based on the teachings and guidance presented herein.

Muteins in accordance with the present invention include proteinsencoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNAor RNA, which encodes the IL-18 inhibitor, in accordance with thepresent invention, under moderately or highly stringent conditions. Theterm “stringent conditions” refers to hybridization and subsequentwashing conditions, which those of ordinary skill in the artconventionally refer to as “stringent”. See Ausubel et al., CurrentProtocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4(1987, 1992), and Sambrook et al. (Sambrook, J. C., Fritsch, E. F., andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washingconditions 12-20° C. below the calculated Tm of the hybrid under studyin, e.g., 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinaryskill in this art understand that stringency conditions also depend onthe length of the DNA sequences, oligonucleotide probes (such as 10-40bases) or mixed oligonucleotide probes. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC.See Ausubel, supra.

In a preferred embodiment, any such mutein has at least 40% identity orhomology with the sequence of SEQ ID NO: 1, 2 or 3 of the annexedsequence listing. More preferably, it has at least 50%, at least 60%, atleast 70%, at least 80% or, most preferably, at least 90% identity orhomology thereto.

Identity reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotides or two polypeptide sequences, respectively, overthe length of the sequences being compared.

For sequences where there is not an exact correspondence, a “% identity”may be determined. In general, the two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orvery similar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. Thus for instance, programs available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux J et al.,1984), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identityand the % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of Smith and Waterman (1981) and finds thebest single region of similarity between two sequences. Other programsfor determining identity and/or similarity between sequences are alsoknown in the art, for instance the BLAST family of programs (Altschul SF et al, 1990, Altschul S F et al, 1997, accessible through the homepage of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990;Pearson 1988).

Preferred changes for muteins in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of IL-18BP polypeptides or proteins or viralIL-18BPs, may include synonymous amino acids within a group which havesufficiently similar physicochemical properties that substitutionbetween members of the group will preserve the biological function ofthe molecule (Grantham, 1974). It is clear that insertions and deletionsof amino acids may also be made in the above-defined sequences withoutaltering their function, particularly if the insertions or deletionsonly involve a few amino acids, e.g., under thirty, and preferably underten, and do not remove or displace amino acids which are critical to afunctional conformation, e.g., cysteine residues. Proteins and muteinsproduced by such deletions and/or insertions come within the purview ofthe present invention.

Preferably, the synonymous amino acid groups are those defined inTable 1. More preferably, the synonymous amino acid groups are thosedefined in Table 2; and most preferably the synonymous amino acid groupsare those defined in Table 3.

TABLE 1 Preferred Groups of Synonymous Amino Acids Amino Acid SynonymousGroup Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe,Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His,Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val GlyAla, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met,Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser,Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr,Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu,Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu,Met Trp Trp

TABLE 2 More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE 3 Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of IL-18BP polypeptides or proteins, ormuteins of viral IL-18BPs, for use in the present invention include anyknown method steps, such as presented in U.S. Pat. Nos. 4,959,314,4,588,585 and 4,737,462, to Mark et al; U.S. Pat. No. 5,116,943 to Kothset al., U.S. Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111to Chong et al; and U.S. Pat. No. 5,017,691 to Lee et al; and lysinesubstituted proteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).

The term “fused protein” refers to a polypeptide comprising an IL-18BP,or a viral IL-18BP, or a mutein or fragment thereof, fused with anotherprotein, which, e.g., has an extended residence time in body fluids. AnIL-18BP or a viral IL-18BP, may thus be fused to another protein,polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.

“Functional derivatives” as used herein cover derivatives of IL-18BPs ora viral IL-18BP, and their muteins and fused proteins, which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e. they do not destroy the activity of the protein whichis substantially similar to the activity of IL-18BP, or viral IL-18BPs,and do not confer toxic properties on compositions containing it.

These derivatives may, for example, include polyethylene glycolside-chains, which may mask antigenic sites and extend the residence ofan IL-18BP or a viral IL-18BP in body fluids. Other derivatives includealiphatic esters of the carboxyl groups, amides of the carboxyl groupsby reaction with ammonia or with primary or secondary amines, N-acylderivatives of free amino groups of the amino acid residues formed withacyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acylderivatives of free hydroxyl groups (for example that of seryl orthreonyl residues) formed with acyl moieties.

As “active fractions” of an IL-18BP, or a viral IL-18BP, muteins andfused proteins, the present invention covers any fragment or precursorsof the polypeptide chain of the protein molecule alone or together withassociated molecules or residues linked thereto, e.g., sugar orphosphate residues, or aggregates of the protein molecule or the sugarresidues by themselves, provided said fraction has substantially similaractivity to IL-18BP.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of IL-18 inhibitor molecule, oranalogs thereof. Salts of a carboxyl group may be formed by means knownin the art and include inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must retain the biological activity of OPNrelevant to the present invention, i.e., exert a proliferative effect onoligodendrocytes.

In a further preferred embodiment of the invention, the inhibitor ofIL-18 is an IL-18 antibody. Anti-IL-18 antibodies may be polyclonal ormonoclonal, chimeric, humanized, or even fully human. Recombinantantibodies and fragments thereof are characterized by high affinitybinding to IL-18 in vivo and low toxicity. The antibodies which can beused in the invention are characterized by their ability to treatpatients for a period sufficient to have good to excellent regression oralleviation of the pathogenic condition or any symptom or group ofsymptoms related to a pathogenic condition, and a low toxicity.

Neutralizing antibodies are readily raised in animals such as rabbits,goat or mice by immunization with IL-18. Immunized mice are particularlyuseful for providing sources of B cells for the manufacture ofhybridomas, which in turn are cultured to produce large quantities ofanti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobulin constant region is derived from a humanimmunoglobulin molecule. Preferably, both regions and the combinationhave low immunogenicity as routinely determined (Elliott et al., 1994).Humanized antibodies are immunoglobulin molecules created by geneticengineering techniques in which the murine constant regions are replacedwith human counterparts while retaining the murine antigen bindingregions. The resulting mouse-human chimeric antibody preferably havereduced immunogenicity and improved pharmacokinetics in humans (Knightet al., 1993).

Thus, in a further preferred embodiment, IL-18 antibody is a humanizedIL-18 antibody. Preferred examples of humanized anti-IL-18 antibodiesare described in the European Patent Application EP 0 974 600, forexample.

In yet a further preferred embodiment, the IL-18 antibody is fullyhuman. The technology for producing human antibodies is described indetail e.g. in WO00/76310, WO99/53049, U.S. Pat. No. 6,162,963 orAU5336100. Fully human antibodies are preferably recombinant antibodies,produced in transgenic animals, e.g. xenomice, comprising all orportions of functional human Ig loci.

In a highly preferred embodiment of the present invention, the inhibitorof IL-18 is an IL-18BP, or an isoform, a mutein, fused protein,functional derivative, active fraction or circularly permutatedderivative thereof. These isoforms, muteins, fused proteins orfunctional derivatives retain the biological activity of IL-18BP, inparticular the binding to IL-18, and preferably have essentially atleast an activity similar to IL-18BP. Ideally, such proteins have abiological activity which is even increased in comparison to unmodifiedIL-18BP. Preferred active fractions have an activity which is betterthan the activity of IL-18BP, or which have further advantages, like abetter stability or a lower toxicity or immunogenicity, or they areeasier to produce in large quantities, or easier to purify.

The sequences of IL-18BP and its splice variants/isoforms can be takenfrom WO 99/09063 or from Novick et al., 1999, as well as from Kim etal., 2000.

Functional derivatives of IL-18BP may be conjugated to polymers in orderto improve the properties of the protein, such as the stability,half-life, bioavailability, tolerance by the human body, orimmunogenicity. To achieve this goal, the functional derivative maycomprise at least one moiety attached to one or more functional groups,which occur as one or more side chains on the amino acid residues. Sucha functional group may e.g. be Polyethlyenglycol (PEG). PEGylation maybe carried out by known methods, described in WO 92/13095, for example.

Therefore, in a preferred embodiment of the present invention, theinhibitors of IL-18, and in particular the IL-18BP is PEGylated.

In a further preferred embodiment of the invention, the inhibitor ofIL-18 is a fused protein comprising all or part of an IL-18 bindingprotein, which is fused to all or part of an immunoglobulin. The personskilled in the art will understand that the resulting fusion proteinretains the biological activity of IL-18BP, in particular the binding toIL-18. The fusion may be direct, or via a short linker peptide which canbe as short as 1 to 3 amino acid residues in length or longer, forexample, 13 amino acid residues in length. Said linker may be atripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a13-amino acid linker sequence comprisingGlu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced betweenthe IL-18BP sequence and the immunoglobulin sequence. The resultingfusion protein has improved properties, such as an extended residencetime in body fluids (half-life), increased specific activity, increasedexpression level, or the purification of the fusion protein isfacilitated.

In a preferred embodiment, IL-18BP is fused to the constant region of anIg molecule. Preferably, it is fused to heavy chain regions, like theCH2 and CH3 domains of human IgG1, for example. The generation ofspecific fusion proteins comprising IL-18BP and a portion of animmunoglobulin are described in example 11 of WO 99/09063, for example.Other isoforms of Ig molecules are also suitable for the generation offusion proteins according to the present invention, such as isoformsIgG₂ or IgG₄, or other Ig classes, like IgM or IgA, for example. Fusionproteins may be monomeric or multimeric, hetero- or homomultimeric.

Interferons are predominantly known for inhibitory effects on viralreplication and cellular proliferation. Interferon-γ, for example, playsan important role in promoting immune and inflammatory responses.Interferon β (IFN-β, an interferon type I), is said to play ananti-inflammatory role.

The invention therefore also relates to the use of a combination of aninhibitor of IL-18 and an interferon in the manufacture of a medicamentfor the treatment of CNS injury.

Interferons may also be conjugated to polymers in order to improve thestability of the proteins. A conjugate between Interferon β and thepolyol Polyethlyenglycol (PEG) has been described in WO99/55377, forinstance.

In another preferred embodiment of the invention, the interferon isinterferon-β (IFN-β), and more preferably IFN-β1a.

The inhibitor of IL-18 production and/or action is preferably usedsimultaneously, sequentially, or separately with the interferon.

Tumor necrosis factor, has been described in the literature to have bothprotective and toxic effects in brain injury (Shohami et al., 1999). InExample 1 below, TNF injection into mice following severe brain traumaresulted in a significant decrease of IL-18 levels in the brain, thusindicating that TNF may have a beneficial effect on the recovery oftraumatic brain injury. Therefore, a preferred embodiment of theinvention relates to the use of an inhibitor of IL-18 in combinationwith TNF for the preparation of a medicament for treatment and/orprevention of brain injury, for simultaneous, sequential or separateuse.

The combination of IL-18 inhibitors with TNF alpha is preferred inaccordance with the present invention.

In a further preferred embodiment of the invention, the medicamentfurther comprises an anti-inflammatory agent, such as an NSAID(nonsteroidal anti-inflammatory drugs). In a preferred embodiment, aCOX-inhibitor, and most preferably a COX-2 inhibitor, is used incombination with an IL-18 inhibitor. COX-inhibitors are known in theart. Specific COX-2 inhibitors are disclosed in WO 01/00229, forexample. The active components may be used simultaneously, sequentially,or separately.

Oxidative stress, in particular reactive oxygen species (ROS), have beendescribed to play a role in the pathophysiology of brain damage (Shohamiet al., 1997).

Therefore, in a preferred embodiment of the present invention, themedicament—further comprises an antioxidant, for simultaneous,sequential, or separate use. Many antioxidants are known in the art,such as vitamins A, C or E, or 5-aminosalicylic acid, or superoxidedismutase.

In a further preferred embodiment of the present invention, theinhibitor of IL-18 is used in an amount of about 0.001 to 100 mg/kg ofbody weight, or about 0.01 to 10 mg/kg of body weight or about 0.1 to 3mg/kg of body weight or about 1 to 2 mg/kg of body weight.

In yet a further preferred embodiment, the inhibitor of IL-18 is used inan amount of about 0.1 to 1000 μg/kg of body weight or 1 to 100 μg/kg ofbody weight or about 10 to 50 μg/kg of body weight.

The invention further relates to the use of a nucleic acid moleculecomprising the coding sequence of an IL-18 inhibitor, a mutein,functional derivative, or active fraction thereof, in the preparation ofa medicament for the prevention and/or treatment of CNS injury.

Preferably, the nucleic acid molecule further comprises a sequence of anexpression vector, e.g. to use gene therapy for administering the IL-18inhibitor of the invention.

Preferably, the nucleic acid molecule is administered intramuscularly.

In order to treat and/or prevent CNS injury, the gene therapy vectorcomprising the sequence of an inhibitor of IL-18 production and/oraction may be injected directly into the diseased tissue, for example,thus avoiding problems involved in systemic administration of genetherapy vectors, like dilution of the vectors, reaching and targeting ofthe target cells or tissues, and of side effects.

The use of a vector for inducing and/or enhancing the endogenousproduction of an inhibitor of IL-18 in a cell normally silent forexpression of an IL-18 inhibitor, or which expresses amounts of theinhibitor which are not sufficient, are also contemplated according tothe invention for treatment and/or prevention of CNS injury. The vectormay comprise regulatory elements functional in the cells desired toexpress the inhibitor or IL-18. Such regulatory sequences or elementsmay be promoters or enhancers, for example. The regulatory sequence maythen be introduced into the right locus of the genome by homologousrecombination, thus operably linking the regulatory sequence with thegene, the expression of which is required to be induced or enhanced. Thetechnology is usually referred to as “Endogenous Gene Activation” (EGA),and it is described e.g. in WO 91/09955.

It will be understood by the person skilled in the art that it is alsopossible to shut down IL-18 expression directly, without using aninhibitor of IL-18, with the same technique. To do that, a negativeregulation element, like e.g. a silencing element, may be introducedinto the gene locus of IL-18, thus leading to down-regulation orprevention of IL-18 expression. The person skilled in the art willunderstand that such down-regulation or silencing of IL-18 expressionhas the same effect as the use of an IL-18 inhibitor in order to preventand/or treat disease.

The invention further relates to the use of a cell that has beengenetically modified to produce an inhibitor of IL-18 in the manufactureof a medicament for the treatment and/or prevention of CNS injury.

The invention further relates to pharmaceutical compositions,particularly useful for prevention and/or treatment of inflammatory CNSinjury, which comprise a therapeutically effective amount of aninhibitor of IL-18 and/or a therapeutically effective amount of aninterferon and/or a pharmaceutically effective amount of TNF and/or apharmaceutically effective amount of an anti-inflammatory agent and/or apharmaceutically effective amount of an anti-oxidative agent.

As inhibitor of IL-18, the composition may comprise caspase-1inhibitors, antibodies against IL-18, antibodies against any of theIL-18 receptor subunits, inhibitors of the IL-18 signaling pathway,antagonists of IL-18 which compete with IL-18 and block the IL-18receptor, and IL-18 binding proteins, isoforms, muteins, fused proteins,functional derivatives, active fractions or circularly permutatedderivatives thereof having the same activity.

IL-18BP and its isoforms, muteins, fused proteins, functionalderivatives, active fractions or circularly permutated derivatives asdescribed above are the preferred active ingredients of thepharmaceutical compositions.

The interferon comprised in the pharmaceutical composition is preferablyIFN-beta or IFN-alpha.

In yet another preferred embodiment, the pharmaceutical compositioncomprises therapeutically effective amounts of TNF alpha. Thepharmaceutical composition according to the invention may furthercomprise one or more COX-inhibitors.

The definition of “pharmaceutically acceptable” is meant to encompassany carrier, which does not interfere with effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which it is administered. For example, for parenteraladministration, the active protein(s) may be formulated in a unit dosageform for injection in vehicles such as saline, dextrose solution, serumalbumin and Ringer's solution.

The active ingredients of the pharmaceutical composition according tothe invention can be administered to an individual in a variety of ways.The routes of administration include intradermal, transdermal (e.g. inslow release formulations), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, intracranial, epidural, rectal, topical, andintranasal routes. Any other therapeutically efficacious route ofadministration can be used, for example absorption through epithelial orendothelial tissues or by gene therapy wherein a DNA molecule encodingthe active agent is administered to the patient (e.g. via a vector),which causes the active agent to be expressed and secreted in vivo. Inaddition, the protein(s) according to the invention can be administeredtogether with other components of biologically active agents such aspharmaceutically acceptable surfactants, excipients, carriers, diluentsand vehicles.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, the active protein(s) can be formulated as a solution,suspension, emulsion or lyophilized powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e.g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

The bioavailability of the active protein(s) according to the inventioncan also be ameliorated by using conjugation procedures which increasethe half-life of the molecule in the human body, for example linking themolecule to polyethylenglycol, as described in the PCT PatentApplication WO 92/13095.

The therapeutically effective amounts of the active protein(s) will be afunction of many variables, including the type of antagonist, theaffinity of the antagonist for IL-18, any residual cytotoxic activityexhibited by the antagonists, the route of administration, the clinicalcondition of the patient (including the desirability of maintaining anon-toxic level of endogenous IL-18 activity).

A “therapeutically effective amount” is such that when administered, theIL-18 inhibitor results in inhibition of the biological activity ofIL-18. The dosage administered, as single or multiple doses, to anindividual will vary depending upon a variety of factors, includingIL-18 inhibitor pharmacokinetic properties, the route of administration,patient conditions and characteristics (sex, age, body weight, health,size), extent of symptoms, concurrent treatments, frequency of treatmentand the effect desired. Adjustment and manipulation of establisheddosage ranges are well within the ability of those skilled in the art,as well as in vitro and in vivo methods of determining the inhibition ofIL-18 in an individual.

According to the invention, the inhibitor of IL-18 is used in an amountof about 0.0001 to 100 mg/kg or about 0.01 to 10 mg/kg or body weight,or about 0.1 to 5 mg/kg of body weight or about 1 to 3 mg/kg of bodyweight or about 1 to 2 mg/kg of body weight. Alternatively, the IL-18inhibitors may be administered in amounts of about 0.1 to 1000 μg/kg ofbody weight or about 1 to 100 μg/kg of body weight or about 10 to 50μg/kg of body weight

The route of administration, which is preferred according to theinvention is administration by subcutaneous route. Intramuscularadministration is further preferred according to the invention. In orderto administer the IL-18 inhibitor directly to the place of its action,it is also preferred to administer it via the intracranial orintrathecal route. The intracranial route is especially preferred incombination with open head injury (missile injury of the brain).

In further preferred embodiments, the inhibitor of IL-18 is administereddaily or every other day.

The daily doses are usually given in divided doses or in sustainedrelease form effective to obtain the desired results. Second orsubsequent administrations can be performed at a dosage which is thesame, less than or greater than the initial or previous doseadministered to the individual. A second or subsequent administrationcan be administered during or prior to onset of the disease.

According to the invention, the IL-18 inhibitor can be administeredprophylactically or therapeutically to an individual prior to,simultaneously or sequentially with other therapeutic regimens or agents(e.g. multiple drug regimens), in a therapeutically effective amount, inparticular with an interferon and/or a TNF and/or anotheranti-inflammatory agent, such as a COX inhibitor and/or an antioxidant.Depending on the brain injury, the co-administration of a TNF-antagonistinstead of TNF itself can also be conceived (Shohami et al., 1999).Active agents that are administered simultaneously with othertherapeutic agents can be administered in the same or differentcompositions.

The invention further relates to a method for the preparation of apharmaceutical composition comprising admixing an effective amount of anIL-18 inhibitor and/or an interferon and/or a TNF antagonist and/or aCOX inhibitor with a pharmaceutically acceptable carrier.

The invention further relates to a method of treatment of CNS injury,comprising administering a pharmaceutically effective amount of an IL-18inhibitor to a patient in need thereof.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents or any other references, are entirely incorporated byreference herein, including all data, tables, figures and text presentedin the cited references. Additionally, the entire contents of thereferences cited within the references cited herein are also entirelyincorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplication such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning an range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES Materials and Methods

Trauma Model

The mice used in this study were males of age 8-16 weeks weighing 30-35g. They were bred in a specific pathogen-free environment, kept understandard conditions of temperature and light in cages of 4-6 animals,and fed with food and water ad libitum. The study was performedaccording to the guidelines of the Institutional Animal Care Committeeof the Hebrew University of Jerusalem, Israel. Experimental CHI wasperformed using the weight-drop device previously developed (Chen et al.1996). Briefly, after induction of ether anesthesia, a midlinelongitudinal incision was performed, the skin was retracted and theskull exposed. The left anterior frontal area was identified and atipped teflon cone was placed ˜1 mm lateral to the midline, in themid-coronal plane. The head was fixed and a 75 g weight was dropped onthe cone from a height of 18 cm, resulting in a focal injury to the lefthemisphere. After trauma, the mice received supporting oxygenation with100% O₂ for no longer than 2 min, and were then brought back to theircages.

Assessment of IL-18 Levels in Mouse Brains

For quantification of intracranial IL-18 levels, mice of the C57BL/6(B6) strain (total n=62) were assigned to six distinct groups: (1)“normal controls”; untreated B6 mice (n=10). (2) “ether anesthesia”;mice were anesthetized with ether for 10 minutes and decapitated after24 h (n=10) or 7 days (n=10). (3) “sham operation”; these mice underwentanesthesia and longitudinal scalp incision and were sacrificed after 24h (n=15) or 7 days. (4) “trauma group”; experimental CHI was performedas described above, and the animals were decapitated in ether anesthesiaat 4 h (n=7), 24 h (n=7), and 7 days (n=7) after trauma. (5) “TNFinjection”; for assessment of a possible role of TNF in the regulationof intracerebral IL-18, mice were ether anesthetized, injectedintra-cerebro-ventricularly (i.c.v.) with 200 ng murine recombinant TNF(R&D Systems, Abingdon, UK) in 10 μl sterile phosphate-buffered saline(PBS) and sacrificed after 24 h (n=10). (6) “mock injection”; theseanimals were injected i.c.v. with vehicle only (10 μl sterile PBS) andsacrificed after 24 h (n=6), as a control group to the TNF-injectedanimals. In all mice, the brains were immediately removed afterdecapitation, snap-frozen in liquid nitrogen and stored at −70° C. untilanalysis. Brains from the trauma group were separated into left(ipsilateral) and right (contralateral) hemisphere, in order to allow acomparison of IL-18 levels in the injured vs. non-injured hemisphere.Tissue homogenization was performed with a Polytron (Kinematica, Kriens,Switzerland) using a dilution of 1:4 in ice cold extraction buffer (W/W)containing Tris 50 mM (pH 7.2), NaCI 150 mM, Triton-X-100 1% (BoehringerMannheim, Rotkreuz, Switzerland), and protease inhibitor cocktail(Boehringer Mannheim). The homogenate was shaken on ice for 90 min andthen centrifuged for 15 min at 3,000 g and 4° C. The supernatants werealiquoted and stored at −70° C. until analysis. The concentrations oftotal protein in the brain extracts were measured by Bradford assay (BioRad Laboratories, Munich, Germany) and found to be very constant in allmice assessed (12.1±2.1 mg/ml; mean±SD). Quantification of intracerebralcytokine levels was performed by ELISA specific for murine IL-18,according to the manufacturer's instructions (R&D Systems, Abingdon,UK). The sensitivity of the assay was 5 pg/ml. For comparison of theintracerebral IL-18 levels between the different animal groups, allconcentrations below the detection limit of 5 pg/ml were assigned avalue of 4.9 pg/mI. The samples were run undiluted in duplicate wellsand the final concentration was calculated from the mean OD of duplicatesamples. The OD was determined by spectrophotometer (DynatechLaboratories Inc., Chantilly, Va.) at an extinction wavelength of 405nm.

IL-18BP Treatment Protocol

Male Sabra mice of the Hebrew University strain (n=40) were used for theIL-18BP studies. Anesthesia and experimental CHI were performed asdescribed above. For the treatment protocol, the animals were dividedinto two groups: In group A (“control group”, n=16), mice were subjectedto experimental CHI, injected with vehicle alone (PBS) after one hour,and observed for 7 days for neurological assessment (see below). Ingroup B (“study group”, n=18), the mice were injected i.p. with 50 μgIL-18BP immediately after determination of the neurological score at t=hafter CHI. Since the blood-brain barrier permeability is 5-6-foldincreased between 14 h post CHI, as previously determined in the sameexperimental model (Chen et al. 1996), IL-18BP is available to the brainunder these conditions. Two additional groups of mice were treatedaccording to groups A and B (group C: “control group”; group D: “studygroup”, respectively) and decapitated after 48 h, followed by braindissection for evaluation of posttraumatic edema, as described below.

Evaluation of Neurological Impairment

For assessment of posttraumatic neurological impairment, a NeurologicalSeverity Score (NSS) has been previously developed and validated (Stahelet al., 2000).

The score consists of 10 individual clinical tasks on motor function,alertness, and physiological behavior, whereby one point is given forfailure of the task and zero points for succeeding (Table 4). A maximalNSS of 10 points indicates severe neurological dysfunction, with failureof all tasks, whereas a score of zero is achieved by healthy uninjuredmice. The NSS at 1 hour after trauma reflects the initial severity ofinjury and is highly correlated with clinical outcome (Beni-Adani et al.2001). Evaluation of task performance was performed by two investigatorswho were blinded about the study groups at the time-points 1 h, 24 h, 72h, and 7 days after experimental CHI. The ΔNSS, calculated as thedifference between NSS at t=1 h and the NSS at any later time-point, isa parameter which reflects the degree of spontaneous recovery followingbrain injury, as described earlier (Chen et al. 1996).

TABLE 4 Neurological Severity Score (NSS) for head-injured mice. Pointsfor Task Description success/fail Exit circle Ability and initiative toexit a circle 0/1 of 30 cm diameter within 3 minutes. Mono-/ Paresis ofupper and/or lower limb of 0/1 Hemiparesis the contralateral side.Straight walk Alertness, initiative, and motor ability 0/1 to walkstraight. Startle reflex Innate reflex; the mouse will bounce in 0/1response to a loud hand clap. Seeking behavior Physiological behavior asa sign of 0/1 “interest” in the environment. Beam balancing Ability tobalance on a beam of 7 mm 0/1 width for at least 10 seconds. Round stickAbility to balance on a round stick of 0/1 balancing 5 mm diameter forat least 10 seconds. Beam walk: 3 cm Ability to cross a 30 cm long beam0/1 of 3 cm width. Beam walk: 2 cm Same task, increased difficulty on0/1 a 2 cm wide beam. Beam walk: 1 cm Idem, increased difficulty on a 10/1 cm wide beam. Maximal score 10

Assessment of Brain Edema

The extent of cerebral edema was evaluated by determining the tissuewater content in the injured hemisphere, as previously described (Chenet al. 1996). Briefly, mice were anesthetized as described above at 48 hafter trauma, which corresponds to a time-point at which edema is stillsignificant in this model system (Chen et al. 1996). After decapitation,the cerebellum and brainstem were removed and a cortical segment of ˜20mg, from an area bordering the trauma site and from it contralateralhemisphere was prepared. The right (non-injured) hemisphere was used asan internal control. The tissue slices were weighed and dried for 24 hat 95° C. After weighing the “dried” sections, the percentage of brainwater content was calculated as:%H₂O=[(wet weight−dry weight)×100]/wet weight.

Brain Injury Patients

Ten patients with isolated severe CHI (mean age±SD: 37±10 years; range24-57 years; 9 males and one female), admitted to the Trauma Division ofthe University Hospital Zurich, were included in this study. Allpatients had a Glasgow Coma Scale (GCS) score≦8 after cardiopulmonaryresuscitation (Teasdale and Jennett, 1974). Following CT scanevaluation, all patients received intraventricular catheters fortherapeutic CSF drainage when the intracranial pressure (ICP) exceeded15 mmHg. No patient was treated with steroids. Patients with multipleinjuries requiring interventions for concomitant thoracic, abdominal,pelvic, spinal injuries, or long bone fractures were excluded from thestudy. The individual outcome was assessed using, the Glasgow OutcomeScale (GOS) (Jennett and Bond, 1975). The protocol for CSF and serumcollection was approved by the Ethics Board Committee of the UniversityHospital, Zurich.

Sample Collection and IL-18 Analysis

The CSF and matched serum samples of CHI patients (n=10) were collecteddaily at one fixed time-point. Control CSF was collected from patientsundergoing diagnostic spinal tap (n=5). These patients did not havesigns of inflammatory CNS disease, based on normal CSF values ofprotein, glucose, and cell count (data not shown). In the CHI group,sample collection was performed for 10 days after trauma, unless theventricular catheter was removed earlier, e.g. in cases where the ICPremained in a normal range (≦15 mmHg) for more than 24 hours. A total of106 matched CSF and serum samples were collected in the trauma patientsanalyzed in this study. All samples were immediately centrifuged aftercollection, aliquoted and frozen at −70° C. until analysis.Quantification of IL-18 levels in CSF and serum was performed by ELISAspecific for human IL-18 using commercially available kits (R&D Systems,Abingdon, UK). As for the murine assay, the sensitivity of the ELISA was5 pg/ml, and the final IL-18 concentration was calculated from the meanOD determined in duplicate samples at an extinction wavelength of 405nm. For comparison of the IL-18 CSF levels between CHI patients andcontrols, all concentrations below the detection limit of 5 pg/ml wereassigned a value of 4.9 pg/ml.

Data Analysis

Statistical analysis of the data was performed on commercially availablesoftware (SPSS 9.0 for Windows™). The non-parametric Mann-Whitney-U testwas used for analysis of data which were not normally distributed, suchas the neurological scores (NSS and ΔNSS). The unpaired Student's t-testwas used for comparison of intracerebral IL-18 concentrations in thedifferent mouse groups and for analysis of differences in brain watercontent in the IL-18BP-treated vs. vehicle-injected mice. The comparisonof human IL-18 levels, either in daily CSF vs. matched serum samples inCHI patients, or in trauma vs. control CSF, were determined using thegeneral linear model for repeated measures ANOVA. A p-value<0.05 wasconsidered to be statistically significant.

Results Example 1 Intracerebral IL-18 Levels in Mice

As shown in FIG. 1, IL-18 was detectable by ELISA in brain homogenatesof untreated (“normal”) control mice of the B6 strain (n=10), with amean level of 27.7±1.7 [±SEM] ng/ml. In the experimental groups, theinduction of ether anesthesia alone or in combination with “sham”operation (i.e. ether anesthesia and longitudinal scalp incision)resulted in significantly elevated intracranial IL-18 levels of 48.9±1.1ng/ml (“ether” group, n=8) and 54.3±2.7 ng/ml (“sham” group, n=13),respectively (p<0.01 vs. “normal” mice, unpaired Student's t-test; FIG.1). The difference between the “ether” and “sham”-treated animals wasnot statistically significant (p=0.16).

In the trauma group (n=21), induction of CHI resulted in elevated IL-18levels both in the injured and in the contralateral hemisphere within 4h (60.6±3.3 and 59.8±5.0 ng/ml, respectively) to 24 h (56.9±2.1 and56.3±3.7 ng/ml, respectively) after trauma, however, the levels were notsignificantly higher compared to the “ether” or “sham” groups (p>0.05).

In contrast to this, by 7 days after CHI, a significant increase of theintracerebral IL-18 concentration was detected in the injuredhemisphere, as compared to ether-anesthetized or sham-operated animals(67.6±5.1 ng/ml vs. 42.2±0.8 and 45.2±0.5 ng/ml, respectively; p<0.01),whereas the IL-18 levels in the contralateral hemisphere were notsignificantly elevated above these two control groups (63.2±6.0 ng/ml;p=0.06).

In order to assess the role of TNF, a crucial mediator of inflammationin this trauma model (Shohami et al. 1999), with regard to theregulation of intracerebral IL-18 levels, an additional group of B6 mice(n=10) were injected i.c.v. with 200 ng murine recombinant TNF in 10 μlsterile PBS and sacrificed after 24 h. As shown in FIG. 1, “mock”injection with vehicle only (n=6) resulted in a significantup-regulation of intracerebral IL-18 within 24 h, as compared tountreated normal B6 mice (53.6±3.9 vs. 27.7±1.7 ng/ml; p<0.001).

The injection of TNF induced a significant attenuation of IL-18 levelsin the intracranial compartment within 24 h (22.1±6.9 ng/ml; n=10), ascompared to the “moc”-injected control group 24 h (53.6±3.9 ng/ml;p<0.001). The IL-18 levels in the “TNF group” were even lower than inuntreated normal mice (27.7±1.7 ng/ml), although in this case thedifference was not statistically significant (p=0.45).

Example 2 Effect of IL-18BP Treatment on the Neurological Recovery AfterTrauma

In order to investigate the hypothesis that inhibition of IL-18 mightfacilitate recovery in brain injury, recovery at different time pointsafter a single injection of IL-18BP was compared. It was previouslyshown that the Neurological Severity Score (NSS) at 1 h following traumareflects most accurately the magnitude of the trauma and correlates withthe volume of the injured tissue as seen in MRI and in histology.

In order to obtain groups of animals with comparable trauma, mice wereassigned to different treatment groups after their initial NSS wasevaluated at t=1 h. As shown in FIG. 2 (NSS in IL-18BP (squares) vs.Control (circles) both groups had a similar initial NSS(1 h)(7.69±0.3023 and 7.44±0.3627 control and IL-18BP respectively)indicating comparable severity of injury.

Evaluation of NSS at later times (1-7 days) revealed that animalstreated intraperitoneally (i.p.) with IL-18BP exhibit considerably lessneurological damage, as evident by NSS values, that reached significanceat 7 days post trauma (p=0.045).

The rate of recovery, expressed as ΔNSS (t)=NSS (1 h)−NSS (t), wascalculated. A higher value of ΔNSS reflects greater recovery and a zeroor negative ΔNSS reflects no recovery or worsening. FIG. 3 depicts theΔNSS values of the two groups. At two time points, both at 24 h and at7d the difference between the average ΔNSS values reached significance.

Another experiment was carried out to investigate whether treatment withIL-18BP, given at 3 days post injury can be effective. The issue oftiming of treatment is critical, and so far therapy was shown to beeffective when given beyond a few hours. Since it had been found thatIL-18 itself rises at 7d post-trauma, and treatment with IL-18BP givenat 1 h post-trauma led to greatest effect also at day 7, it was decidednow to treat the mice at day 3 post injury. For comparison, anothergroup was treated at 1 h and at 3d with IL-18BP. The control group wastreated with the solvent (vehicle) only.

The results of this experiment are depicted in FIG. 4, from which it isclear that a single treatment given at day 3 is as effective as thatgiven one hour after CHI and again at 3 days.

This experiment demonstrates the dramatic beneficial effect of aone-time administration of IL-18BP, either given 1 h or 3 days afterclosed head injury, on recovery from traumatic head injury in anexperimental murine model.

Example 3 Elevated IL-18 Levels in Human CSF after Brain Injury

IL-18 levels were assessed in daily CSF and serum samples from 10patients with severe CHI for up to 10 days after trauma. The patients'demographic and clinical data are presented in Table 5.

TABLE 5 Demographic and clinical data of patients with severe CHI^(a)IL-18 in CSF^(d) IL-18 in serum Age (years)/ (pg/ml) (pg/ml) Patient No.gender GCS^(b) GOS^(c) median [range]^(e) median [range]^(e) 1 48/m 3 1283  [78-966] 57.5  [12-66] 2 31/m 7 4 228.4  [22-745] 19.7 [4.9-108] 326/m 5 3 208.5  [20-392] 37.2  [14-163] 4 57/m 5 4 72.6  [30-286] 48.9 [14-104] 5 24/m 4 5 32.6 [4.9-155] 17 [4.9-58] 6 36/m 8 1 49.4 [10-290] 16.7  [12-67] 7 38/f 3 4 17.9  [11-100] 13 [4.9-46] 8 35/m 3 369.8 [4.9-329] 19.8   [7-77] 9 37/m 3 4 37  [23-75] 57.3  [25-98] 1041/m 7 5 4.9 [4.9-169] 26.2  [15-38] Control CSF (n = 5) 4.9 [4.9-7.8]^(a)CHI, closed head injury ^(b)GCS, Glasgow Coma Score (Teasdale andJennett, 1974). ^(c)GOS = Glasgow Outcome Score at 3 months afterinjury; 5 = asymptomatic, 4 = moderate disability, 3 = severedisability, 2 = persistant vegetative state, 1 = death (Jennett andBond, 1975). ^(d)CSF = cerebrospinal fluid. ^(e)IL-18 levels below thedetection limit of 5 pg/ml were assigned a value of 4.9 pg/ml.

As shown in Table 5, the intrathecal IL-18 levels were significantlyelevated in 9/10 CHI patients, as compared to control CSF from 5patients without trauma or inflammatory neurological disease (p<0.05;repeated measures ANOVA). Only one patient #10) had IL-18 CSF levels,which were not significantly elevated as compared to control CSF(p=0.31). The median levels and individual ranges of IL-18 in CSF andserum are presented in Table 5.

Notably, the maximal IL-18 concentrations in CSF (966 ng/ml) were up to200-fold higher in head-injured patients than in controls. IntracerebralIL-18 was detectable by ELISA in 90% of all CSF samples in the traumagroup, whereas only 40% control CSF samples had detectable intracerebralIL-18 levels (i.e. >4.9 ng/ml). In 8/10 CHI patients, the median IL-18concentrations were significantly higher in CSF than in serum (p<0.05;repeated measures ANOVA). However, in two patients (#9, 10) the medianIL-18 levels in serum exceed the corresponding concentrations in CSF, asshown in Table 5.

These results show that there are significantly elevated levels of IL-18in the cerebrospinal fluid of traumatic head injury patients. Theaddition of IL-18BP may reduce these elevated levels, and may thus exertits beneficial effect on recovery from closed head injury, as shown inExample 2 above.

REFERENCES

-   1. Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul S F    et al, Nucleic Acids Res., 25:389-3402, 1997-   2. Chater, K. F. et al., in “Sixth International Symposium on    Actinomycetales Biology”, Akademiai Kaido, Budapest, Hungary (1986),    pp. 45-54).-   3. Chen, Y., S. Constantini, V. Trembovler, M. Weinstock, and E.    Shohami. 1996. An experimental model of closed head injury in mice:    pathophysiology, histopathology, and cognitive deficits. J.    Neurotrauma 13:557-68-   4. Conti, C. B., N.Y. Calingasan, Y. Kim, H. Kim, Y. Bae, E. Gibson,    and T. H. Joh. 1999. Cultures of astrocytes and microglia express    interleukin-18. Mol. Brain Res. 67:46-52-   5. Conti, B., J. W. Jahng, C. Tinti, J. H. Son, and T. H. Joh. 1997.    Induction of interferon-gamma inducing factor in the adrenal    cortex. J. Biol. Chem. 272:2035-2037.-   6. Culhane, A. C, M. D. Hall, N. J. Rothwell, and G. N.    Luheshi. 1998. Cloning of rat brain interleukin-18 cDNA. Mol.    Psychiatry 3:362-6    -   a. Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984.-   7. DiDonato, J A, Hayakawa, M, Rothwarf, D M, Zandi, E and Karin, M.    (1997), Nature 388, 16514-16517.-   8. Elliott, M. J., Maini, R. N., Feldmann, M., Long-Fox, A.,    Charles, P., Bijl, H., and Woody, J. N., 1994, Lancet 344,    1125-1127.-   9. Fassbender, K., O. Mielke, T. Bertsch, F. Muehlhauser, M.    Hennerici, M. Kurimoto, and S. Rossol. 1999. Interferonγ-inducing    factor (IL-18) and interferon-γ in inflammatory CNS diseases.    Neurology 53:1104-6-   10. Jander, S., and G. Stoll. 1998. Differential induction of    interleukin-12, interleukin-18, and interleukin-1β coverting enzyme    mRNA in experimental autoimmune encephalomyelitis of the Lewis    rat. J. Neuroimmunol. 91:93-9-   11. Lancet 1975 Mar. 1; 1(7905):480-4. Jenneft B, Bond M.-   12. Izaki, K. (1978) Jpn. J. Bacteriol. 33:729-742).-   13. Kim S H, Eisenstein M, Reznikov L, Fantuzzi G, Novick D,    Rubinstein M, Dinarello C A. Structural requirements of six    naturally occurring isoforms of the IL-18 binding protein to inhibit    IL-18. Proc Natl Acad Sci U S A 2000; 97:1190-1195.-   14. Kossmann, T., P. F. Stahel, P. M. Lenzlinger, H. Redl, R. W.    Dubs, O. Trentz, G. Schlag, and M. C. Morganti-Kossmann. 1997.    Interleukin-8 released into the cerebrospinal fluid after brain    injury is associated with blood brain-barrier dysfunction and nerve    growth factor production. J. Cereb. Blood Flow Metab. 17:280-9-   15. Knight D M, Trinh H, Le J, Siegel S, Shealy D, McDonough M,    Scallon B, Moore M A, Vilcek J, Daddona P, et al. Construction and    initial characterization of a mouse-human chimeric anti-TNF    antibody. Mol Immunol 1993 Nov. 30: 16 1443-53-   16. Maliszewski, C. R., T. A. Sato, T. Vanden Bos, S. Waugh, S. K.    Dower, J. Slack, M. P. Beckmann, and K. H. Grabstein. 1990. Cytokine    receptors and B cell functions. I. Recombinant soluble receptors    specifically inhibit IL-1- and IL-4-induced B cell activities in    vitro. J. Immunol. 144:3028-3033.-   17. Micallef, M. J., T. Ohtsuki, K. Kohno, F. Tanabe, S. Ushio, M.    Namba, T. Tanimoto, K. Torigoe, M. Fujii, M. Ikeda, S. Fukuda,    and M. Kurimoto. 1996. Interferon-gamma-inducing factor enhances T    helper 1 cytokine production by stimulated human T cells: synergism    with interleukin-12 for interferon-gamma production. Eur-J-Immunol    26:1647-51 issn: 0014-2980.-   18. Morganti-Kossmann, M. C., P. M. Lenzlinger, V. Hans, P.    Stahel, E. Csuka, E. Ammann, R. Stocker, O. Trentz, and T.    Kossmann. 1997. Production of cytokines following brain injury:    beneficial and deleterious for the damaged tissue. Mol. Psychiatry    2:133-6-   19. Nakamura K, Okamura H, Wada M, Nagata K, Tamura T. Infect Immun    1989 February; 57(2):590-5-   20. Novick, D, Kim, S-H, Fantuzzi, G, Reznikov, L, Dinarello, C, and    Rubinstein, M (1999). Immunity 10, 127-136.-   21. Okamura H, Nagata K, Komatsu T, Tanirn-oto T, Nukata Y, Tanabe    F, Akita K, Torigoe K, Okura T, Fukuda S, et al. Infect Immun 1995    October; 63(10):3966-72-   22. Parnet, P, Garka, K E, Bonnert, T P, Dower, S K, and Sims, J E.    (1996), J. Biol. Chem. 271, 3967-3970.    -   a. Pearson W R, Methods in Enzymology, 183, 63-99, 1990    -   b. Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,        2444-2448, 1988-   23. Prinz, M., and U. K. Hanisch. 1999. Murine microglial cells    produce and respond to interleukin-18. J. Neurochem. 72:2215-8-   24. J Clin Invest 1997 Feb. 1; 99(3):469-74 Rothe H, Jenkins N A,    Copeland N G, Kolb H.-   25. Scherbel, U., R. Raghupathi, M. Nakamura, K. E. Saatman, J. Q.    Trojanowski, E. Neugebauer, M. W. Marino, and T. K. Mcintosh. 1999.    Differential acute and chronic responses of tumor necrosis    factor-deficient mice to experimental brain injury. Proc. Natl.    Acad. Sci. USA 96:8721-6-   26. Shohami, E., I. Ginis, and J. M. Hallenbeck. 1999. Dual role of    tumor necrosis factor alpha in brain injury. Cytokine Growth Factor    Rev. 10:119-30-   27. Shohami, E; Beit-Yannai E., Horowitz M; Kohen R (1997): J.    Cereb. Blood Flow Metab. 17, 1007-1019.-   28. Teasdale G, Jennett B. Lancet 1974 Jul. 13; 2(7872):81-4-   29. Stahel P F, Shohami E, Younis F M, Kariya K, Otto V I,    Lenzlinger P M, Grosjean M B, Eugster H P, Trentz O, Kossmann T,    Morganti-Kossmann M C. J Cereb Blood Flow Metab 2000 February;    20(2):369-80-   30. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe    F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S,    Ikeda M, Okamura H, Kurimoto M. J Immunol 1996 Jun. 1;    156(11):4274-9-   31. Wheeler, R. D., A. C. Culhane, M. D. Hall, S.    Pickering-Brown, N. J. Rothwell, and G. N. Luheshi. 2000. Detection    of the interleukin-18 family in rat brain by RT-PCR. Mol. Brain Res.    77:290-3-   32. Whalen, M. J., T. M. Carlos, P. M. Kochanek, S. R.    Wisniewski, M. J. Bell, R. S. Clark, S. T. DeKosky, D. W. Marion,    and P. D. Adelson. 2000. Interleukin-8 is increased in cerebrospinal    fluid of children with severe head injury. Crit. Care Med. 28:929-34-   33. Yoshimoto T, Takeda, K, Tanaka, T, Ohkusu, K, Kashiwamura, S,    Okamura, H, Akira, S and Nakanishi, K (1998), J. Immunol. 161,    3400-3407.

1. A method of treatment of an isolated closed head injury comprisingadministering to an individual in need thereof an effective inhibitingamount of IL-18BP, wherein the IL-18BP is administered in a single doseat 3 days after the closed head injury.
 2. A method for the treatment ofan isolated closed head injury comprising administering to an individualin need thereof an effective amount of IL-18BP and a pharmaceuticallyacceptable carrier, and wherein the IL-18BP is administered in a singledose at 3 days after the closed head injury.
 3. The method of claim 1 or2, wherein the IL-18BP inhibitor is used in an amount of about 0.01 to10 mg/kg of body weight.
 4. The method of claim 1 or 2, wherein theIL-18BP inhibitor is used in an amount of about 0.01 to 10 mg/kg of bodyweight.
 5. The method of claim 1 or 2, wherein the IL-18BP inhibitor isused in an amount of about 0.1 to 5 mg/kg body weight.
 6. The method ofclaim 1 or 2, wherein the IL-18BP inhibitor is used in an amount ofabout 1 to 3 mg/kg of body weight.
 7. The method according to claim 1 or2, wherein the IL-18BP inhibitor is administered subcutaneously.
 8. Themethod according to claim 2, wherein the IL-18BP is glycosylated atleast at one site.
 9. The method according to claim 2, wherein theIL-18BP comprises an immunoglobulin (Ig) fusion.
 10. The methodaccording to claim 2, wherein the IL-18BP comprises at least one moietyattached to at least one functional group which occur as one or moreside chains on the amino acid residues, wherein the moiety is apolyethylene glycol (PEG).
 11. The method according to claim 2, furthercomprising administering to the individual in need thereofsimultaneously, sequentially, or separately an anti-inflammatory agent.12. The method according to claim 11, wherein the anti-inflammatoryagent is a COX-inhibitor.
 13. The method according to claim 2, furthercomprising administering to the individual in need thereofsimultaneously, sequentially, or separately an antioxidant.